Conserved mechanisms of ciliary signaling and cell-cell fusion

纤毛信号传导和细胞间融合的保守机制

• 批准号: 10707152
• 负责人: William J Snell
• 金额: $ 56.16万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707152
• 关键词: Adenylate Cyclase; Adhesions; Affect; Africa; Apical; Automobile Driving; Binding; Binding Proteins; Biochemical; Biological; Biological Models; Cell Adhesion Molecules; Cell Communication; Cell Fractionation; Cell Wall; Cell fusion; Cell membrane; Cell-Cell Adhesion; Cells; Chimeric Proteins; Chlamydomonas; Chlamydomonas reinhardtii; Cilia; Communication; Complement; Concentration Camps; Coupling; Culicidae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Cytoplasm; Dengue; Development; Disease; Dissection; Erinaceidae; Event; Evolution; Fertilization; Foundations; G-Protein-Coupled Receptors; Germ Cells; Green Algae; Hand; Homologous Gene; Human; Huntingtin-Associated protein 1; Image; Immunofluorescence Immunologic; Laboratory Study; Learning; Ligands; Link; Lipid Bilayers; Location; Malaria; Mating Types; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microtubules; Modeling; Molecular; Organelles; Organism; PDAP2 Gene; Partner in relationship; Pathway interactions; Peptides; Phosphorylation; Plasmodium; Process; Property; Protein Family; Protein Kinase; Protein S; Proteins; Reaction; Regulation; Rest; Signal Pathway; Signal Transduction; Site; Sperm-Ovum Interactions; Structure; System; Time; Vertebrates; Vesicle; Virus; ZIKA; adhesion receptor; body system; ciliopathy; erection; field study; malaria transmission; malaria transmission-blocking vaccine; male; patched protein; protein function; receptor; response; restraint; trafficking; transmission process

We propose to use fertilization in the green alga Chlamydomonas as a model system to investigate conserved but still poorly understood cellular and molecular mechanisms of ciliary signaling and the gamete membrane fusion reaction. When Chlamydomonas gametes of opposite mating types are mixed together they adhere to each other by complementary adhesion receptors on their cilia. Interactions between the adhesion receptors SAG1 on plus gametes and SAD1 on minus gametes initiate a signaling pathway that is transmitted to the cell body and triggers a rapid increase in cellular cAMP that activates the gamete for cell-cell fusion. Regulation of ciiliary cyclic nucleotides by ciliary receptor engagement has been conserved throughout evolution and in almost all ciliary signaling events in vertebrates, the primary response to engagement of ciliary receptor is a change in the concentration of cAMP within the organelles. Moreover, in most vertebrate ciliary systems, the mechanisms that transmit the increase in ciliary cAMP to a cellular response are unclear. We have discovered that a protein kinase, GSPK, in Chlamydomonas functions at a previously unrecognized step in ciliary signaling. GSPK is localized in the cytoplasm, not the cilia, yet the entire cellular complement of GSPK is phosphorylated within 1 minute after engagement of the ciliary adhesion receptors. Furthermore, GSPK phosphorylation is upstream of the cAMP increase and GSPK function is required for the rapid increase in cAMP. Our findings set the stage for new strategies to investigate cellular and molecular mechanisms that couple ciliary receptor engagement to a cytoplasmic response. We will use a combination of cell biological, biochemical, and imaging strategies to investigate the mechanisms underlying GSPK function. Gamete activation also activates erection of apically localized protrusions, the plus and minus mating structures, from the plasma membrane of each gamete that are the sites of membrane fusion during fertilization..We have made exciting advances in understanding conserved molecular mechanisms of the gamete membrane fusion reaction, and we now know more about cell- cell fusion in Chlamydomonas than is known in any other system. 1) We showed that bilayer merger of the two cells is driven by trimer formation of eukaryotic class II fusion protein, HAP2, a ancient protein required for fertilization in organisms across kingdoms. 2) We have now identified the separate cell-cell adhesion proteins s FUS1 and MAR1 at the fusion site of each cell. 3) We demonstrated that the adhesion protein MAR1 on minus gametes not only binds to FUS1 on plus gametes, but MAR1 is also biochemically and functionally linked to HAP2. 4) We showed that membrane adhesion mediated by FUS1-MAR1 interactions releases the restrained, prefusion form of HAP2, leading to the irreversible structural rearrangements into the fusion-driving HAP2 trimer. 5) In collaborative laboratory studies and field studies in Africa, we showed that targeting the conserved fusion loop of Plasmodium HAP2 inhibits the mosquito transmission of malaria. Our discoveries now make possible a detailed, structure- and function-based molecular dissection of ciliary signaling and gamete fusion..

我们建议使用绿藻衣藻的受精作为模型系统来研究保守的 但对纤毛信号传导和配子膜的细胞和分子机制仍知之甚少 聚变反应。当相反交配类型的衣藻配子混合在一起时，它们会粘附 通过纤毛上的互补粘附受体相互连接。粘附受体之间的相互作用 正配子上的 SAG1 和负配子上的 SAD1 启动传输至细胞的信号通路 体内并触发细胞 cAMP 快速增加，从而激活配子进行细胞-细胞融合。监管 通过纤毛受体参与的纤毛环核苷酸在整个进化过程中一直是保守的，并且几乎在 在脊椎动物的所有纤毛信号事件中，对纤毛受体参与的主要反应是 细胞器内 cAMP 的浓度。此外，在大多数脊椎动物的睫状系统中，该机制 将纤毛 cAMP 的增加传递给细胞反应的机制尚不清楚。我们发现有一种蛋白质 衣藻中的激酶 GSPK 在纤毛信号传导中以前未被识别的步骤中发挥作用。 GSPK 是 定位于细胞质，而不是纤毛，但 GSPK 的整个细胞补体在 1 内被磷酸化 纤毛粘附受体接合后一分钟。此外，GSPK 磷酸化位于 cAMP的快速增加需要cAMP的增加和GSPK功能。我们的研究结果为 研究将纤毛受体与纤毛受体结合的细胞和分子机制的新策略 细胞质反应。我们将结合细胞生物学、生化和成像策略来 研究 GSPK 功能的潜在机制。配子激活也激活顶端的勃起 每个配子质膜上的局部突出物，即正负交配结构 是受精过程中膜融合的部位。我们在理解方面取得了令人兴奋的进展 配子膜融合反应的保守分子机制，我们现在对细胞了解更多 衣藻中的细胞融合比任何其他系统中已知的都要多。 1）我们证明了两者的双层合并 细胞由真核 II 类融合蛋白 HAP2 的三聚体形成驱动，HAP2 是一种古老的蛋白质， 各个王国的生物体受精。 2）我们现在已经鉴定出单独的细胞-细胞粘附蛋白 FUS1 和 MAR1 位于每个细胞的融合位点。 3）我们证明了负号上的粘附蛋白MAR1 配子不仅与正配子上的 FUS1 结合，而且 MAR1 也在生化和功能上与 HAP2。 4）我们表明，FUS1-MAR1相互作用介导的膜粘附释放了受限制的， HAP2 的预融合形式，导致不可逆的结构重排成融合驱动的 HAP2 三聚体。 5）在非洲的合作实验室研究和实地研究中，我们表明针对保守融合 Plasmodium HAP2 环抑制疟疾的蚊子传播。我们的发现现在使 对纤毛信号传导和配子融合进行详细的、基于结构和功能的分子解剖。